Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a

Abstract

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

“…Our data also show that AcrIIA14 does not interfere with SauCas9 binding to either RNA or dsDNA, but inhibits dsDNA cleavage activity at a potency comparable to AcrIIA13 and AcrIIA15. It is possible that AcrIIA14 either causes the SauCas9-sgRNA RNP to form higher order structures like AcrVA4 (33) or AcrIIC3 (31,32), or interacts with RNP to prevent cleavage but not binding like AcrIIC1 (31). We also observed potent and SauCas9-specific inhibition by AcrIIA13 and AcrIIA14 in mammalian cells (Fig, 4).…”

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

“…To date, inhibition of target binding is the prevalent strategy. Thirteen anti-CRISPR proteins interfere with target recognition and binding (type I-F AcrIF1, AcrIF2 and AcrIF10 [34][35][36][37]; type II-A AcrIIA2, AcrIIA4, AcrIIA5 and AcrIIA6 [38][39][40][41][42][43][44][45][46][47]; type II-C AcrIIC3, AcrIIC4 and AcrIIC5 [48][49][50][51]; type V-A AcrVA1, AcrV4A and AcrVA5 [52][53][54][55][56][57][58]), while only five-block target cleavage (type I-E AcrIE1 [29,59]; type III-B AcrIIIB1 [12]; type I-F AcrIF3 [26,27]; type II-C AcrIIC1 and AcrIIC3 [48][49][50]) ( Figure 1). Given that DNA binding is the ratelimiting step of Cascade and Cas9-mediated interference activities [60,61], altering this step is, therefore, an efficient way to inactivate CRISPR-Cas interference.…”

“…AcrIIA6 and AcrVA4 both function as dimers that tightly associate with a mixed protein-RNA region distinct from the DNA-binding crevasse and catalytic domains [47,54,57,58]. However, regions of both subunits that compose the AcrIIA6 dimer form each binding interface, while every subunit of the AcrV4A dimer contains one binding interface ( Figure 2B).…”

“…Additionally, AcrVA4 can also associate with DNA-bound effector complexes. When it binds to the LbCas12a-crRNA-dsDNA complex with a complete R-loop, it induces the release of the bound DNA before cleavage [54,58]. When it binds to the post-cleavage Cas12a-crRNA-dsDNA complex, it likely blocks the recycling of the enzyme [58].…”

Machine Learning Predicts New Anti-CRISPR Proteins

“…However, a weakness of all of these methods is that they are unable to predict a priori whether a gene may be an Acr, largely because Acr proteins do not share high sequence similarity or mechanisms of action (14,16,(29)(30)(31)(32)(33)(34)(35). One theory to explain the high diversity of Acrs is the rapid mutation rate of the mobile genetic elements they are found in and the need to evolve with the co-evolving CRISPR-Cas systems trying to evade anti-CRISPR activity.…”